Cell-free tissue replacement for tissue engineering

ABSTRACT

The present invention is a natural, cell-free tissue replacement that does not require difficult or extensive preparation made by washing tissue replacement in a solution including one or more sulfobetaines and an anionic surface-active detergent and washing the tissue replacement in serial solutions of the buffered salt to remove excess detergent. The natural, cell-free tissue replacement may be a nerve graft that supports axonal regeneration, guides the axons toward the distal nerve end and/or is immunologically tolerated. Other forms of the invention are a composition and kit prepared by the method of making a native, cell-free tissue replacement. The present invention may be modified for use in diagnostic, therapeutic, and prophylactic applications.

This application claims priority to pending provisional patentapplication Ser. No. 60/414,278, filed Sep. 27, 2002.

The U.S. Government may own certain rights in this invention pursuant tothe terms of the National Science Foundation Grant No. BES 9733156 andNational Institute of Health Training Grant No. 732 GM 08474-09.

FIELD OF THE INVENTION

The present invention relates generally to the field of tissueengineering and specifically to the use of cell-free tissue replacementsfor promoting tissue regeneration.

BACKGROUND OF THE INVENTION

Tissue engineering faces several challenges in the development ofreplacement tissue. First, the “replaced” tissue must promote tissueregeneration. In doing so, the replaced tissue must be compatible withthe tissue it is replacing so that neighboring cells accept thereplacement and do not disrupt tissue continuity. Importantly, thereplaced tissue also has to overcome the immunologic challenges faced byany new addition to a biologic system, that of a “foreign body.”Furthermore, to be successful, the replaced tissue must eventuallyexhibit the properties and function of tissue that it is replacing. Forexample, the replaced tissue should exhibit similar mechanical andstructural properties of the native environment or at a minimum, notinterfere with the native environment. The replaced tissue may also actas a scaffold to promote cellular regeneration. Finally, the replacedtissue should not stimulate scar formation that limits tissueregeneration or inhibits the natural function of the underlying tissue.

Strategies for successful regeneration include the use of biologic orbiocompatible materials to build a bridge across the injured area. Whilesuccessful for some tissue, many biomaterials have been rejected or havepromoted regional “scarring.” In addition, the mechanical and structuraldifferences that define the function of different types of tissue haveproven difficult to overcome, especially for tissue such as nerves.

These same strategies are modeled on the fabrication of synthetic orbiocompatible tissue in vitro that is representative of a native tissue.One example is the use of vascular grafts using an acellular tubularstructure that is then implanted at the injured site. The grafts willeventually be invaded by normal cells and the tubular structure willremain viable over time. While promising for tissue with limited needsfor mobility, these biocompatible structures are generally stiffer thanthe surrounding tissue and uncompromising in areas requiringflexibility. Alternatively, biodegradable scaffolds have been engineeredwith the hope that, over time, the degradable component(s) will bereplaced by constituents that make up the normal tissue and will exertthe same function as performed by the original tissue. The biodegradablescaffold strategy has seen limited success except to accelerateotherwise naturally occurring phenomena, and have not successfullyreplaced tissue with high structural or mobility requirements (e.g.,bone, nerve, muscle).

For some tissue (e.g. nerve tissue), several other techniques have beenused to try to initiate tissue regeneration. For example, acuteadministration of hydrophilic polymer or polymer blends is used to sealnerve membranes. The polymer application may reconnect or fuse severedaxons of damaged nerve membranes and may even promote recovery ofexcitability in some damaged nerve fibers. However, large injuries areineffectively repaired using this method, where tension has been foundto limit regeneration.

Peripheral nerve allografts have shown some promising success,generally, in the presence of one or more immunosuppressive agents toreduce nerve rejection. The popularity of using nerve conduits fortissue regeneration has increased recently due to the need foralternative tissue reconstruction techniques that yield fewercomplications and greater mobility for the individual. In fact, activeregenerating fibers on a proximal stump of a nerve have been found toregenerate and progress as a fascicular unit in optimum condition at thetrunk of another healthy nerve.

There remains a need to improve tissue replacements, especially fortissue such as nerve that has proven difficult to regenerate withcurrent tissue replacement strategies. The improved tissue replacementshould maintain native characteristics of the tissue it is replacing, beable to incorporate bioactive compounds or molecules where necessary topromote rapid regeneration, and stimulate tissue repair and regenerationin the absence of tissue scarring that reduces tissue mobility andintegrity. Despite current research efforts in tissue regeneration,there still exists a need for a clinically attractive alternative toautografts.

SUMMARY OF THE INVENTION

The subject matter of the present invention includes a novel method andcomposition for a replacement tissue that has undergonedecellularization while retaining its native structure and integrity. Bypreserving the structure of the acellular replacement tissue, lessremodeling is required by the host after implantation. The presentinventors recognized that the native tissue itself serves as the mostphysiologic environment for tissue regeneration, that is, native tissueappears to be the most viable option for healthy regeneration. To repaira nerve over a gap, e.g., autologous nerve grafts are used herein tophysically guide the regenerating axons and to prevent the infiltrationof occluding connective tissue. The present invention overcomes severaldisadvantages of current art, namely, loss of function at the donorsite, mismatch of nerve cable dimensions between the donor graft andrecipient nerve, and the need for multiple surgeries.

The present invention is a natural replacement tissue or graft notrequiring difficult preparation steps with varying temperatures,radiation and/or harsh chemical treatments. The compositions and methodsof the invention specifically remove the cellular components withoutsignificantly altering the natural extracellular structure. The nativeextracellular matrix (ECM) structure is preserved (referred to as intactstructural components), specifically, the basal laminae andendoneurium/endothelial layer retain their natural and generallyoriginal structure. In one embodiment, the invention includes anoptimized acellular nerve graft that supports axonal regeneration,guides the axons toward the distal nerve end and is toleratedimmunologically. The optimized acellular nerve graft may be, e.g., anisograft, an autograft, an allograft and even a xenograft.

In one form, the present invention is a method for preparing a native,cell-free tissue replacement that includes the steps of; washing thetissue replacement overnight in a solution comprising sulfobetaines,washing tissue replacement in serial solutions of a buffered salt,washing tissue replacement in a mixture of sulfobetaines with an anionicdetergent, e.g., Triton X-200, and washing the tissue replacement inserial solutions of the buffered salt to remove excess detergent.

The present invention is also a native, cell-free tissue replacement,e.g., made using the method of the present invention. In another form,the present invention is a kit for tissue replacement that a cell-freenative tissue replacement. The kit may also include one or moresolutions useful for re-suspending the cell-free native tissuereplacement of the present invention, e.g., a buffered, sterile salinesolution that is pharmacologically acceptable as will be know to theskilled artisan. Furthermore, the native, cell-free tissue replacementor graft may also include a vial or solution with cells that may beadded to gaps to improve the growth of nerve and other cells, e.g.,Schwann cells. The kit may further include an instruction sheet orbooklet that provides the user with detailed instructions for the useand procedure for insertion of the native, cell-free tissue replacement.In one embodiment, the native, cell-free tissue replacement forms partof a suture, tube, sheet, film, scaffold, valve, limb replacement,tissue transplant, and joint for delivery into a host.

The present invention is also an optimized acellular graft that supportsaxonal regeneration, guides the axons toward the distal nerve end and isimmunologically tolerated. In one example, the graft is a nerve graft.The acellular graft may also include a cell, a polymer, a bioactivecompound or combinations thereof and may be stored at a low temperature,e.g., about 0 to 4 degrees centigrade in a sterile, buffered solutionuntil use. The temperature may be lower or higher depending on thesolution in which the graft is stored before use, e.g., including one ormore preservative agents. In one embodiment, the acellular graft mayalso include one or more cells placed in the gap between prior to graftimplantation. The graft of the present invention is able to be implantedwith reduced T-cell mediated immune response, e.g., CD8+ or CD4+ Tcells.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and further advantages of the invention may be betterunderstood by referring to the following description in conjunction withthe accompanying figures in which:

FIG. 1 depicts a flowchart for the preparation of the present invention;

FIG. 2 is a graph that summarizes a tissue immune response with CD8+invasion in accordance with the present invention;

FIG. 3 is a graph showing the tissue immune response with macrophageinvasion in accordance with the present invention;

FIGS. 4A, 4B, 4C and 4D are longitudinal sections of tissue that werecut from (4A) fresh isografts, (4B) fresh allografts, (4C) optimizedacellular isografts, and (4D) optimized acellular allografts that wereharvested 28 days after implantation (Scale bar=200 μm);

FIG. 5 is a graph that summarizes the cell-mediated immune response inthe fresh and acellular nerve grafts was evaluated by determining thepercentage of tissue covered by CD8+ cells.

FIGS. 6A, 6B, 6C and 6D are longitudinal sections of tissue were cutfrom (6A) fresh isografts, (6B) fresh allografts, (6C) optimizedacellular isografts, and (6D) optimized acellular allografts that wereharvested 28 days after implantation (Scale bar=200 μm);

FIG. 7 is a graph that summarizes the level of macrophages present infresh and acellular nerve grafts after 28 days was evaluated bydetermining the percentage of area stained in longitudinal tissuesections;

FIGS. 8A, 8B and 8C show the Axonal regeneration through acellular nervegrafts was demonstrated by staining longitudinal tissue segments forneurofilaments. The random patterns in the axons at the junctions of theproximal nerve and graft (8A) and the graft and distal nerve (8C)suggest a lack of guidance as the axons crossed into and out of thegraft (Scale bar=100 μm);

FIGS. 9A, 9B, 9C and 9D are cross-sections of basal laminae werevisualized by the staining of laminin protein, the ring-like appearanceof the open tubes in (9A) fresh nerve tissue, (9B) tissue decellularizedwith the optimized protocol, and (9C) tissue decellularized with thefreeze/thaw protocol (Scale bar=10 μm); and

FIGS. 10A and 10B are graphs that show the regenerative capacity of fournerve graft models was evaluated by measuring axon density incross-sections of the grafts (10A) 28 days after implantation and (10B)84 days after implantation.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed herein in terms of a biomaterial for use as atissue replacement (e.g., peripheral nerve graft, a tissue graft, orimplant that can be used to study and stimulate growth andregeneration), a conducting polymer that can used to stimulate cellresponse, and method for preparing the biomaterial, it should beappreciated that the present invention provides many inventive conceptswhich can be embodied in a wide variety of specific contexts. Thespecific embodiments discussed herein are merely illustrative of ways tomake and use the invention are not meant to limit the scope of thepresent invention in any way.

Terms used herein have meanings as commonly understood by a person ofordinary skill in the areas relevant to the present invention. Termssuch as “a,” “an,” and “the” are not intended to refer to only asingular entity, but include the general class of which a specificexample may be used for illustration. The terminology herein is used todescribe specific embodiments of the invention, but their usage does notlimit the invention, except as outlined in the claims. All technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs, unless defined otherwise. Methods and materials similar orequivalent to those described herein may be used in the practice ortesting of the present invention, the generally used methods andmaterials are now described. The following abbreviations are usedherein: extracellular matrix (ECM), sulfobetaine-10 (SB-10),sulfobetaine-16 (SB-16), 3,3′ diaminobenzidine (DAB), HarlanSprague-Dawley (HSD), horseradish peroxidase (HRP), majorhistocompatibility complex (MHC) and phosphate buffered saline (PBS).

There is currently no effective treatment for damage to central nervoussystem (CNS) nerves or for absolute tissue regeneration, although drugscan reduce swelling and damage to tissue such as the spinal cord. Incontrast to spinal cord injury, there exist therapies, although notoptimal, for the treatment of damaged peripheral tissue such as nerves.Current clinical treatments for peripheral nerve injury are surgicalend-to-end anastomoses and autologous nerve grafts. Surgical end-to-endrepair involves the direct reconnection of individual nerve fasciclesand is useful only if nerve ends are directly adjacent, as tension inthe nerve cable prohibits regeneration.

A replacement is needed for the autologous graft, the primary clinicaltreatment of peripheral nerve injuries that involve a defect too largeto be repaired by end-to-end reconnection. As described hereinabove, thepresent inventors have developed an optimized acellular nerve allograftthat retains the extracellular structure of peripheral nerve tissue viaan improved chemical decellularization treatment. The decellularizationprocess removes cellular membranes from nerve tissue, thus eliminatingthe antigens responsible for allograft rejection.

The present invention describes the composition and method of anatural-based biomaterial graft developed from native tissue that isfree of cells while retaining the intrinsic structure and architectureof the native tissue. As used herein the term “native, cell-free tissuereplacement” is used to describe a tissue that has been removed from ahost animal (live or cadaveric) that has been treated according to thepresent invention (also referred to as “acellular grafts”). The term“optimized acellular nerve allograft,” as used herein is used todescribe a native, nerve cell-free tissue replacement allograft. As usedherein, the terms “cell-free,” “acellular,” “decellular,” are usedinterchangeably and all represent a tissue generally absent of cells,e.g., living cells. The removal or absence of cells will depend on thedetergents and conditions used for removal of cells. The level of cellremoval will depend on the exact source, methodology and need for theremoval of cells. By cell removal a broad range of removal may be usedwith the present invention, e.g., removal of 70, 80, 90, 95, 99 or about100 percent of living cells is included. The extent of cell removalreduces the likelihood of a strong immunologic rejection when using anon-autologous tissue source or a source of tissue that is not matchedin terms of histocompatibility, e.g., from a donor from the same or evena different species (a xenograft).

The term “acellular graft” as used herein refers to biological materialderived from a donor for transplantation into a recipient. The graft isderived from any mammalian source, including human, whether fromcadavers or living donors. In some cases it is useful that the source ofthe graft donor is matched for HLA class I and/or class II antigens withthe recipient or host.

The term “mammal” refers to any animal host classified as a mammal,including humans, domestic and farm animals, and zoo, sports, or petanimals, such as dogs, horses, cats, cows, etc. By “compatible” is meanta mammalian host that will accept the donated graft, e.g., a human host.If both the donor of the graft and the host are human, they arepreferably matched for HLA class II antigens so as to improvehistocompatibility. The present invention, however, serves to overcomeproblems with problems generally associated with lack ofhistocompatibility by providing a tissue replacement that does notstimulate the host immune response.

The term “donor” as used herein refers to the mammalian species, dead oralive, from which the graft is derived, e.g. a human donor. Human donorsmay be volunteers that are blood-related donors that are normal onphysical examination and of the same major ABO blood group, becausecrossing major blood group barriers possibly prejudices survival of theallograft.

The term “transplant” and variations thereof refers to the insertion ofa graft into a host, whether the transplantation is syngeneic (where thedonor and recipient are genetically identical), allogeneic (where thedonor and recipient are of different genetic origins but of the samespecies), or xenogeneic (where the donor and recipient are fromdifferent species). Thus, in a typical scenario, the host is human andthe graft is an isograft, i.e., derived from a human of the same ordifferent genetic origins. The graft, herein referred to as native,cell-free tissue replacement tissue, may be used as a simplified modelsystem with which to study the various stages and critical factorsinvolved in tissue regeneration. In addition, the model native,cell-free tissue replacement tissue may be used for clinicalapplications. The replacement tissue is created generally from humancadaver tissue, however, other tissue sources such as from the host or adonor, be it human or other mammal, may be used.

An optimized combination of detergents in a salt and phosphate bufferedsolution are used in methods of the present invention. The detergentsinclude sulfobetaines with a hydrophilic tail composed of 10 to 16carbons and Triton X-200. Detergents used in the present invention arethose that specifically rupture cells inside the tissue but do notdamage the structural proteins (those that include the extracellularmatrix). Cellular debris may be removed using a number of techniquesknown in the art, such as washing with buffered isotonic solutionsand/or physically removing other non-structural debris such as fat.Compared to an allograft, the resulting acellular replacement tissueelicits a significantly reduced immunologic response because surfacecell antigens have been removed.

Several uses of the present invention include: 1) as biocompatiblebiomaterial that is generally non-immunogenic; 2) as a structuralfoundation for other soluble factors to promote tissue- andhost-specific, tissue regeneration; and 3) as a research tool to studythe host response to replacement tissue structure. The present inventionalso includes a replacement tissue that is a native, generally acellulartissue with processible, biologic, bioactive, and/or biodegradablefeatures. Preparation of the present invention and examples are furtherprovided below.

Tissue Replacement Compositions and Delivery. The present invention maybe delivered to the body in the form of a modified native, cell-freetissue replacement structure in the form of, e.g., sutures, tubes,sheets, films, valves, joints, vessels, and scaffolds. These modifiednative, cell-free tissue replacement structures (also referred herein asscaffolds) are thus prepared using the methods taught herein andmodified as discussed below. For example, the native, cell-free tissuereplacement may be cast using procedures known to those skilled in theart.

While cells are generally removed from the present invention, it may beacceptable and often necessary to reintroduce one or more different typeof cells to the present invention. These cells may be obtained directfrom a donor, from a culture of cells from a donor, or from cellculture. Donor cells are generally obtained by biopsy and grown toconfluence in culture using standard conditions apparent to those ofskill in the art. The donor or cells obtained from the donor may beimmunosuppressed as needed, for example, using a schedule of steroidsand other immunosuppressant drugs, if required. Immunosuppression of thehost may provide immunoprotection of cell transplants while a new tissueor tissue equivalent is growing by excluding the host immune system. Inaddition, the present invention may be used to provide multiple celltypes, including genetically altered cells, clones or transplants,within the three-dimensional architecture of the present invention forthe purpose of transplant engraftment, immunotherapy, cognitivefunction, tissue regeneration, repair or reconstruction. Examples ofcells include, but are not limited to, chondrocyte, osteoblast, musclecell, thyroid cell, parathyroid cell, immune cell, pancreatic cell,fibroblast, hepatocyte, epithelial cell, islet cell, nerve cell, andother cells acting primarily to synthesize and secrete or metabolizematerials, as well as biopsied or cloned cells of the intestines,kidney, heart, brain, spinal cord, muscle, skeleton, liver, stomach,skin, lung, reproductive system, nervous system, immune system, spleen,bone marrow, lymph nodes, glands.

Alternatively, the present invention may include bioactive moleculesformulated with one or more active species so that the present inventionbecomes a carrier for one or more active species. Whether native orcasted, the native, cell-free tissue replacement may also include activeagents incorporated into an added polymer or polymer solution (e.g., apolymer scaffold) or may be attached directly to the surface of orwithin the present invention using techniques readily apparent to thoseskilled in the art. For example, the active agents may be added bycuring on and into the native, cell-free tissue replacement, bondedionically, covalently and/or using a crosslinking agent, e.g., acleavable cross-linking agent.

In some instances, it may be useful to incorporate or attach theinactive version of the active agent or species (active agent andspecies may be used interchangeably) that can then be activated to theactive species as needed and required, possibly in a time-releasedmanner, light-activated (e.g., UV) activated in situ by local enzymes orby providing the site with an activating agent exogenously, e.g.,providing a patient with an oral activating agent. The active agent maybe a drug or other biologically active compound; thus the presentinvention may be a microcarrier for the delivery of drugs or otherbiologically active compounds when used in the body. Examples ofbiologically active compounds are proteins, peptides, polysaccharides,nucleic acids, oligonucleotides, natural and synthetic organic orinorganic molecules, and those biologic molecules used for therapeutic,prophylactic or diagnostic purposes. Drugs may include antibiotics,antivirals, chemotherapeutic agents, immunosuppressive agents, growthfactors, anti-angiogenic agents, hormones, anti-inflammatory agents,drugs having an effect on vascular flow, cellular metabolics, or thatare effective against one or more diseases and/or combinations thereof.

Other active agents may also be included with the present invention,e.g., non-steroidal anti-inflammatory drugs (NSAIDs) such as propionicacid derivatives; acetic acid derivatives; fenamic acid derivatives;biphenylcarboxylic acid derivatives; and oxicams. Examples of propionicacid derivatives include: ibuprofen, naproxen, ketoprofen, flurbiprofen,fenoprofen, suprofen, fenbufen, and fluprofen may be mentioned aspreferred compounds. Acetic acid derivatives derivatives include:tolmetin sodium, zomepirac, sulindac and indomethacin. Fenamic acidderivatives derivatives include: mefenamic acid and meclofenamatesodium. Diflunisal and flufenisal are biphenylcarboxylic acidderivatives, while oxicams include piroxicam, sudoxicam and isoxicam.Other analgesics for use with the present invention includeacetominophen and phenacetin. Those skilled in the art will appreciatethat any of the foregoing compounds may be used in the form of theirpharmaceutically acceptable salt forms, e.g. carboxylic acids withpotassium or sodium counter-ions, and the like. The present inventionmay, therefore, be selectively combined with cells and/or bioactivecompounds (e.g., those with active species) to promote tissue/limbreconstruction, tissue regeneration, or tissue/cell/limb transplantationand engraftment. For example, the present invention with cells may becombined with one or more active species such as angiogenic factors,antibiotics, anti-inflammatories, growth factors, alone or incombination with other compounds that induce differentiation and/or celland tissue growth and regeneration.

Other additives conventionally used in implantable compositions may beincluded, which are well known in the art. Such additives include,e.g.,: anti-adherents (anti-sticking agents, glidants, flow promoters,lubricants) such as talc, magnesium stearate, fumed silica), micronizedsilica, polyethylene glycols, surfactants, waxes, stearic acid, stearicacid salts, stearic acid derivatives, starch, hydrogenated vegetableoils, sodium benzoate, sodium acetate, leucine, PEG-4000 and magnesiumlauryl sulfate.

Other additives include, binders (adhesives), i.e., agents that impartcohesive properties to powdered materials through particle-particlebonding, such as matrix binders (dry starch, dry sugars), film binders(PVP, starch paste, celluloses, bentonite and sucrose), and chemicalbinders (polymeric cellulose derivatives, such as carboxy methylcellulose, HPC and HPMC; sugar syrups; corn syrup; water solublepolysaccharides such as acacia, tragacanth, guar and alginates; gelatin;gelatin hydrolysate; agar; sucrose; dextrose; and non-cellulosicbinders, such as PVP, PEG, vinyl pyrrolidone copolymers, pregelatinizedstarch, sorbitol, and glucose).

For certain implanted composition that also include active agents it maybe useful to provide buffering agents (or bufferants), where the acid isa pharmaceutically acceptable acid, such as hydrochloric acid,hydrobromic acid, hydriodic acid, sulfuric acid, nitric acid, boricacid, phosphoric acid, acetic acid, acrylic acid, adipic acid, alginicacid, alkanesulfonic acid, amino acids, ascorbic acid, benzoic acid,boric acid, butyric acid, carbonic acid, citric acid, fatty acids,formic acid, fumaric acid, gluconic acid, hydroquinosulfonic acid,isoascorbic acid, lactic acid, maleic acid, methanesulfonic acid, oxalicacid, para-bromophenylsulfonic acid, propionic acid, p-toluenesulfonicacid, salicylic acid, stearic acid, succinic acid, tannic acid, tartaricacid, thioglycolic acid, toluenesulfonic acid and uric acid, and wherethe base is a pharmaceutically acceptable base, such as an amino acid,an amino acid ester, ammonium hydroxide, potassium hydroxide, sodiumhydroxide, sodium hydrogen carbonate, aluminum hydroxide, calciumcarbonate, magnesium hydroxide, magnesium aluminum silicate, syntheticaluminum silicate, synthetic hydrotalcite, magnesium aluminum hydroxide,diisopropylethylamine, ethanolamine, ethylenediamine, triethanolamine,triethylamine, triisopropanolamine, or a salt of a pharmaceuticallyacceptable cation and acetic acid, acrylic acid, adipic acid, alginicacid, alkanesulfonic acid, an amino acid, ascorbic acid, benzoic acid,boric acid, butyric acid, carbonic acid, citric acid, a fatty acid,formic acid, fumaric acid, gluconic acid, hydroquinosulfonic acid,isoascorbic acid, lactic acid, maleic acid, methanesulfonic acid, oxalicacid, para-bromophenylsulfonic acid, propionic acid, p-toluenesulfonicacid, salicylic acid, stearic acid, succinic acid, tannic acid, tartaricacid, thioglycolic acid, toluenesulfonic acid, and uric acid.

In some compositions additives may also include: chelating agents (suchas EDTA and EDTA salts); colorants or opaquants (such as titaniumdioxide, food dyes, lakes, natural vegetable colorants, iron oxides,silicates, sulfates, magnesium hydroxide and aluminum hydroxide);coolants (e.g., trichloroethane, trichloroethylene, dichloromethane,fluorotrichloromethane); cryoprotectants (such as trehelose, phosphates,citric acid, tartaric acid, gelatin, dextran and mannitol); and diluentsor fillers (such as lactose, mannitol, talc, magnesium stearate, sodiumchloride, potassium chloride, citric acid, spray-dried lactose,hydrolyzed starches, directly compressible starch, microcrystallinecellulose, cellulosics, sorbitol, sucrose, sucrose-based materials,calcium sulfate, dibasic calcium phosphate and dextrose).

It should be appreciated that there is considerable overlap between theabove-listed additives in common usage, since a given additive is oftenclassified differently by different practitioners in the field, or iscommonly used for any of several different functions. Thus, theabove-listed additives should be taken as merely exemplary, and notlimiting, of the types of additives that can be included in compositionsof the present invention. The amounts of such additives may be readilydetermined by one skilled in the art, according to the particularproperties desired.

One method of the present invention is a native, cell-free tissuereplacement (with or without cells) that has been at least partiallypurified (in a purifed or inactive form) in a buffered solution asoutlined in the flowchart of FIG. 1. The native, cell-free tissuereplacement purified as demonstrated herein may also include one or moreactive agents may or may not then be trapped within and/or on theinvention by a variety of means, including, but not limited to,cross-linking the active agents to the native, cell-free tissuereplacement of the present invention. Non-adsorbed or trapped molecules(and or cells) are washed off prior to use. Alternatively, the one ormore active agents may be dried directly onto the surface of the presentinvention or may be cross-linked to, e.g., a polymer or saccharide (withor without cells), that is incorporated onto the surface of or withinthe present invention.

For example, one or more active and inactive agents factors may be mixedin a slow-release form with a cell-saccharide suspension and allowed tocontact and permeate the present invention prior to its implant ortransplantation. The saccharide may then be cross-linked to trap thecells within and on the present invention. Alternatively, a polymersolution may be modified to bind the one or more agents or signalrecognition sequences prior to its combination with a suspension ofcells. In one embodiment of the present invention the agents, active orinactive, may include whole, partially purified or purified proteins,saccharides, lipids and the like from tissue humors, e.g., blood, tissueascities, peritoneal lavage and the like, as will be known to theskilled artisan.

The native, cell-free tissue replacement of the present invention mayalso be used for direct implantation and/or injection. When used torepair tissue, promote regeneration, replace damaged cells, enhancegrowth, proliferation and differentiation or for transplantation,reconstruction, and improved tissue, organ or limb function, theeffectiveness of the present invention (including the cells and/oractive species components where applicable) can readily be optimized bythose skilled in the art without undue experimentation by using themethods described herein.

The present invention may be implanted with materials that includesutures, tubes, sheets, adhesion prevention devices, wound healingproducts, tissue healing agents and other tissue or cell growthpromoters that further enhance the effectiveness of tissue regeneration.In addition, when provided with an electrically conductive material avoltage or current may be applied directly to the present invention atthe repair, implant, transplant or reconstruction site. Polymers orother molecules with piezoelectric or electrically conducting propertiesmay also be incorporated into the present invention. Severalelectroactive polymers exist including piezoelectric (e.g.,polyvinylidene fluoride) and electrically conducting materials (e.g.,polypyrrole (PP), and polythiophene). Since piezoelectric materialsdepend on small mechanical deformations to produce transient surfacecharges, the level and duration of focused stimulation cannot becontrolled. In contrast, electrically conducting polymers readily permitexternal control over both the level and duration of stimulation. Thus,strategies designed to enhance the regeneration of a responsive cellmight employ electrically conducting polymers. For diagnostic purposes,the present invention may be incorporated not only with moleculescontaining active species but also with one or more detectable agents ormolecules that allows for the diagnosis, monitoring and/or prophylacticmeasures. Examples of suitable detectable agents include dyes, labels,metals, detection devices, and electronic chips.

Thus, the methods used to prepare the present invention and thecomposition of the present invention serve several beneficial functionswhen used in mammals, including regenerative, restorative,reconstructive, therapeutic, prophylactic, and diagnostic functions.Applications include its use as a graft, implant, scaffold, limbreplacement, in transplantation, muscle or tissue resection, and as atube, sheet, film, vessel or nerve.

EXAMPLE 1 Preparation of Native Cell-free Tissue

Solutions used for the preparation of native, acellular nerve tissueinclude, e.g., RPMI medium, double distilled water (ddH2O), 100 mM Na/50mM phosphate buffered solution, 50 mM Na/10 mM phosphate bufferedsolution, SB-10 (5×CMC) in 50 mM Na/10 mM phosphate buffered buffersolution, an anionic surface-active detergent (such as TritonX-200/SB-16 [0.07%/5×CMC] in 50 mM Na/10 mM phosphate bufferedsolution). All solutions were either filter sterilized or autoclaved asappropriate for the preparation of samples to be used in a sterilefield; techniques of which are readily apparent to those of skill in theart. Examples of anionic surface-active detergents that may be used andadapted with the method of the present invention include, e.g., TRITONX-200, TRITON W-30 Conc., TRITON GR-5M, TRITON GR-7M, TRITON DF-20, andTRITON QS-44.

In advance of retrieving the tissue, sterile RPMI medium is added to,e.g., a 15 mL conical tube and kept on ice. The sciatic nerve tissuefrom a donor animal is generally harvested using sterile techniques.Harvested tissue is immediately placed in cold RPMI medium, preferablyunder a flow hood or in a sterile environment (e.g., operating room),and return tube to ice. Tissue is then arranged for viewing under adissecting microscope in the laminar flow hood, preferably wiped down inadvance with a sterile solution such as 70% ethanol. A dissectingmicroscope may not be necessary for larger tissue that is readily viewedby eye. It will generally be useful to clean the fat, excess blood andother tissue or tissue debris from the tissue to be implanted in asterile environment and while in a nutrient media. Nerve tissue, forexample, may be placed in a sterile Petri dish filled with nutrientmedium such as RPMI. Nerve is best viewed under the microscope to removeexcess fat and connective tissue.

After cleaning the tissue, tissue is placed in a sterile tube containingddH₂O. Again, to maintain sterility, all remaining steps shouldgenerally be performed in a sterile environment such as a laminar flowhood, clean room or other similar sterile field. Tissue is rinsed inddH₂O for about 7 hours with several changes of solution. Solutions maybe removed after each change using a sterile-tipped vacuum aspirator,where the tissue is first centrifuged to keep it at the bottom of thetube. Several additional washing solutions follow, including: SB-10solution, solution overnight; three times in 100 mM Na/50 mM phosphatebuffered solution, washes should be about 15 minutes each; TritonX-200/SB-16 solution for about 24 hours; three times in 100 mM Na/50 mMphosphate buffered solution, washes should be about 15 minutes each;SB-10 solution for about 7 hours; three times in 100 mM Na/50 mMphosphate buffered solution, washes should be about 15 minutes each;Triton X-200/SB-16 solution, overnight; three times in 100 mM Na/50 mMphosphate buffered solution, washes should be about 15 minutes each.Tissue can then be stored at a low temperature, e.g., about 4 degreesCentigrade (4° C.) in 50 mM Na/10 mM phosphate buffered solution untiluse. The compositions and methods disclosed herein allow for the use ofother detergent combinations for the removal of cells without creatingstructural damage (thereby retaining extracellular matrix and essentialcomponents), as will be recognized by the skilled artisan. Detergentsand conditions for the use of the detergents, buffers and ionicconditions that mimic those disclosed herein may be used by the skilledartisan and are encompassed by the present invention.

Immune Response of Native Cell-free Tissue.

The immune response of tissue prepared with the method of the presentinvention show that the native cell-free tissue adapts to itsenvironment and is not rejected. Furthermore, the composition of thepresent invention (the native cell-free tissue) is not rejected as othertissue replacements or allografts typically are. Examples of this arepresented in more detail below.

Native-cell free tissues were evaluated for their immune responsefollowing implantation as a cell-free sciatic nerve graft. Fourconditions were evaluated and include:

-   -   1. Fresh isografts (i.e., not treated with methods of the        present invention) removed/harvested from three Lewis rats        (i.e., the donors) and implanted, one each, into three Lewis        rats (i.e., the hosts);    -   2. Fresh allografts (i.e., not treated with methods of the        present invention) removed/harvested from five Lewis rats (i.e.,        the donors) and implanted, one each, into five Harlan Sprague        Dawley rats (i.e., the hosts);    -   3. Cell-free native tissue allografts (i.e., treated with the        methods of the present invention) removed/harvested from five        Lewis rats (i.e., the donors) and implanted, one each, into five        Harlan Sprague Dawley rats (i.e., the hosts); and    -   4. Cell-free native tissue isografts (i.e., treated with the        methods of the present invention) removed/harvested from three        Sprague Dawley rats (i.e., the donors) and implanted, one each,        into three Harlan Sprague Dawley rats (i.e., the hosts).

Conditions 2 and 4 were used specifically because Lewis and HarlanSprague Dawley rats express different RT1 halotypes, and, in general,are found to reject tissue that is not from the same species (i.e.,Harlan Sprague Dawley reject tissue from Lewis rats and vice versa). Thesame-strain, fresh isograft implant (condition 1) was used to examinethe immune response resulting from implantation of a graft containingviable cells. This condition is largely compatible with the currentclinical approach (the autograft) used for several types of tissuerepair, such as nerve tissue repair. The cross-strain, fresh allograftimplant (condition 2) was used as a typical positive rejection immuneresponse. Condition 3 or the cross-strain, cell-free native tissueallografts were used to determine if immunologic rejection occurredafter using a composition and method of the present invention. Andcondition 4 using the same-strain cell-free native tissue isograft wasdesigned to evaluate immunologic responses after surgery employing thecomposition and methods of the present invention.

Observed immune responses that were followed were CD8+ invasion andmacrophage invasion. Macrophages are immune cells that are released inresponse to injury and believed to be one cell responsible for clearingthe debris that occurs at and near the site of injury. It is believedthat when foreign bodies are introduced at the site of injury, such asduring tissue replacement or grafting, macrophages will invade the sitein even higher numbers and are, thus, involved in clearing the site ofall foreign bodies, including the graft. This is especially true when agraft is rejected.

Rat T-cytotoxic lymphocytes are CD8+ cells, and are also used asindicators of foreign body rejection, including graft or implantrejection. A subset of CD8+ cells are found at a site of injury (e.g.,after sciatic nerve injuries), even in the absence of immunologicrejection. Therefore, macrophages and some CD8+ cells are generallypresent after an implantation, tissue replacement, transplantation orgrafting procedure. On the other hand, a significant increase in CD8+cells in the absence of a large number of macrophages generallyindicates rejection of the implant, replacement, transplant, or graft.

All grafts were implanted in the right sciatic nerve of male rats atleast about 350 g in weight. Rats were allowed to recover for 28 daysand then anesthetized to expose the implanted grafts. Following fixationof each graft from each rat, grafts were extracted, embedded inparaffin, sectioned, sections attached to glass slides, and stained forinvading CD8+ and macrophage cells. Methods used for graft fixation,extraction, paraffin embedding, and sectioning are those readilyapparent to one of ordinary skill in the art of tissue preparation andhistology.

Tissue sections were stained for the presence of macrophages and CD8+cells. Sections were viewed under a microscope, and several images ofeach section were captured with a digital camera. Using image analysissoftware, the percentage of graft tissue that was positively stained foreither macrophages or CD8+ cells was determined.

FIG. 2 is a graph that shows the percentage of CD8+ cells that werepresent after independent grafting using each of the four conditions.FIG. 3 is a graph that shows the percentage of macrophage cells thatwere present after independent grafting using each of the fourconditions. The CD8+ cell invasion results shows that the cross-strain,fresh allograft (condition 2) is undergoing rejection. The levels ofCD8+ cells in the cell-free native tissue grafts (conditions 3 and 4) islower than that observed with the same-strain, fresh isograft (condition1). Therefore, the cell-free native tissue grafts are not eliciting anincreased T-cytotoxic lymphocyte invasion, and thus, not undergoingimmunologic rejection.

Macrophage invasion into the cross-strain, fresh allograft (condition 2)was the highest, as would be expected for a graft undergoing rejection.The two cell-free native tissue grafts (conditions 3 and 4) showed lowermacrophage invasion than the cross-strain, fresh allograft (condition2), but both were higher than the same-strain, fresh graft (condition1), and indicates that an immune response is occurring the cell-freetissue; however the immunologic response is not considered to be arejection, as is evident from the CD8+ stain. The increased level ofmacrophages could be the result of several factors, including the moreopen structure of the cell-free native tissue grafts, compared to themass transfer inhibiting compact nature of fresh grafts.

The most important finding is that the level of CD8+ cells in the twocell-free native tissue grafts (conditions 3 and 4) is on the same orderas that seen in the fresh isografts (condition 1), despite the increasein macrophage levels. Thus, the cell-free native tissue grafts are notundergoing immunological rejection.

TABLE 1 Sect. 1 + Sect. 2 + Sect. 3 + Sect. 4 + Sect. 5 + Avg. % AreaArea Area Area Area Avg. Area % Coverage Coverage Std Dev Condition 13.671 0.922 1 8.37 6.86 10.26 12.18 10.51 9.636 3.778824 2 11.46 12.3610.88 13.42 9.7 11.564 4.534902 3 5.6 5.32 9.06 4.78 9.67 6.886 2.700392Condition 2 8.703 2.133 4 23.39 21.78 22.05 25.12 22.79 23.026 9.0298045 19.76 21.05 23.69 28.14 20.1 22.548 8.842353 9 34.84 22.45 25.2 29.1740.48 30.428 11.93255 12 14.8 17.61 15.3 15.02 16.5 15.846 6.214118 1522.45 18.21 17 19.25 18.67 19.116 7.496471 Condition 3 2.091 0.657 64.23 3.98 5.26 4.79 4.52 4.556 1.786667 7 6.11 5.31 5.29 4.11 5.61 5.2862.072941 8 4.46 4.93 5.32 5.49 4.62 4.964 1.946667 11 6.72 9.59 8.0710.39 5.91 8.136 3.190588 14 3.12 3.65 3.58 3.77 4.44 3.712 1.455686Condition 4 1.574 0.331 13 2.35 3.74 3.3 3.48 3.3 3.234 1.268235 16 3.94.3 4.12 2.68 4.48 3.896 1.527843 17 4.47 6.06 3.65 4.74 5.64 4.9121.926275 Abbreviations are: Sect. = section; Avg. = average; + =positively stained; Std Dev = standard deviation.

Applications of the present invention include the treatment of injuries(surgical or non-surgical and spinal cord or non-spinal cord injuries),in plastic and reconstructive surgery, for transplantation or asimplants, especially in difficult tissue such as peripheral nervetissue.

EXAMPLE 2 Replacement Nerve Tissue Graft

A replacement is needed for the autologous graft, the primary clinicaltreatment of peripheral nerve injuries that involve a defect too largeto be repaired by end-to-end reconnection. As described hereinabove, thepresent inventors have developed an optimized acellular nerve allograftthat retains the extracellular structure of peripheral nerve tissue viaan improved chemical decellularization treatment. The decellularizationprocess removes cellular membranes from nerve tissue, thus eliminatingthe antigens responsible for allograft rejection.

In this example, the optimized acellular grafts were tested in vivo fortheir immunogenicity and regenerative capacity using a well-establishedrat model system for immunological response of nerve tissue transplants.To test the immunogenicity of the acellular grafts, nerve tissue fromHarlan Sprague-Dawley rats was decellularized and implanted into Lewisrats for 28 days. Histological examination of the levels of CD8+ cellsand macrophages that infiltrated the acellular grafts suggested that thedecellularization process averted cell-mediated rejection of the grafts.In a subsequent experiment, regeneration in the optimized grafts after28 and 84 days was compared to that in fresh isografts and two publishedacellular graft models. It was found that using the present invention,the average axon density at the midpoint of the optimized graft wasstatistically indistinguishable from that in the fresh isograft at bothtime points, 96% and 910% higher than in the thermally decellularizedmodel described by Gulati (1988), and 42% and 401% higher than in thechemically decellularized model described by Sondell et al. (1998).Using the present invention, the optimized acellular grafts wereimmunologically tolerated by the removal of cellular material andpreservation of the ECM accomplished with the decellularization processdescribed hereinabove and are both beneficial for promoting regenerationthrough an acellular nerve graft.

Materials and Methods: Creation of Grafts.

To create the optimized acellular grafts, both the left and rightsciatic nerves were harvested under aseptic conditions from 350 g HarlanSprague-Dawley (HSD) male rats. All chemicals were purchased from Sigma(St. Louis, Mo.) unless otherwise noted. All solutions were autoclavedor filter sterilized prior to use. The tissue was handled only by theends to minimize structural damage. When harvested the nerves wereimmediately placed in RPMI 1640 solution at 4° C. All subsequent stepswere conducted in a laminar flow hood for sterility. Fatty andconnective tissue were removed from the nerve epineurium. The nervetissue was cut into 15 mm segments and placed in a 15 ml conical tubefilled with deionized distilled water. All washing steps were carriedout at 25° C. with agitation. After 7 hours, the water was aspirated andreplaced by a solution containing 125 mM sulfobetaine-10 (SB-10), 10 mMphosphate, and 50 mM sodium. The nerves were agitated for 15 hours. Thetissue was then rinsed once for 15 minutes in a washing solution of 50mM phosphate and 100 mM sodium. Next, the washing solution was replacedby a solution containing 0.14% Triton X-200, 0.6 mM sulfobetaine-16(SB-16), 10 mM phosphate, and 50 mM sodium. After agitating for 24hours, the tissue was rinsed with the washing solution 3 times at 5minutes per rinse. The nerve segments were again agitated in the SB-10solution (7 hours), washed once, and agitated in the SB-16/Triton X-200solution (15 hours). Finally, the tissue segments were washed 3 timesfor 15 minutes in a solution containing 10 mM phosphate and 50 mMsodium, then stored in the same solution at 4° C.

Acellular nerve grafts were also created according to previouslyestablished methods as a basis for comparison. The chemicallydecellularized model was created by a protocol published by Sondell, etal. Briefly, the nerve tissue was agitated in distilled water for 7hours, 46 mM Triton X-100 in distilled water overnight, and then 96 mMsodium deoxycholate in distilled water for 24 hours. These steps wererepeated before performing a final wash in distilled water. Alltreatment steps were performed at room temperature and the tissue wassubsequently stored in a 10 mM phosphate buffered saline (PBS) solutionat 4° C.

The thermally decellularized model (i.e., a freeze/thaw graft) wascreated according to the protocol described by Gulati, et al.Immediately after harvest, the nerve tissue was dipped in liquidnitrogen for 20 seconds, thawed in PBS at room temperature for 60seconds, and then the process was repeated four additional times. Thegrafts were placed in PBS at room temperature and used within 30minutes.

Implantation of Grafts.

Isografts and allografts were used to study the immunogenicity of theoptimized acellular grafts. Isografts were harvested from a donor animal(e.g., Lewis rat) of the same strain as the host animal (e.g., Lewisrat); they mimic the autograft. The autografts served as a negativecontrol, as the immune response would only be due to the surgicalprocedure. Allografts were harvested from a donor animal (e.g., HSD rat)of a different strain than the host animal (e.g., Lewis rat). The freshallograft served as a positive control since it elicits a cell-mediatedrejection. The acellular isograft was used to examine the in vivoresponse to the treatment protocol (e.g., the response to residualchemicals).

Table 2 summarizes the test conditions and controls for immunologicaltolerance determined. The optimized acellular graft was inspected forresidual antigens by implanting an acellular allograft. In summary, atotal of four conditions studied were tested: (a) fresh isografts (n=6)(b) fresh allografts (n=5); (c) optimized acellular isografts (n=5); and(d) optimized acellular allografts (n=5) (Table 2).

TABLE 2 Implants to Examine Immunological Tolerance of Optimized GraftsDonor Host Number of Analyzing Response Graft Type Strain StrainImplants to: Fresh Isograft Lewis* Lewis 3 Surgical procedure HSD^(†)HSD 3 (negative control) Fresh Allograft Lewis HSD 5 Natural antigens(positive control) Acellular Isograft HSD HSD 5 Treatment protocolAcellular Lewis HSD 5 Residual antigens Allograft *Lewis rats are aninbred strain (i.e., greater than 98% genetic homogeneity) ^(†)HSD ratsare an outbred strain, but the animals used were from a closed colony.

Each rat was anesthetized with an intra-peritoneal injection of 120mg/kg body weight ketamine (Webster Veterinary Supply, Sterling, Mass.)and 15 mg/kg body weight xylazine (Webster Veterinary Supply). Thesciatic nerve on the right side was exposed, transected, and 5 mm ofnerve was removed. The ends of the graft were trimmed immediately priorto implantation to attain a clean-cut, 10 mm graft. The graft wassutured to both the proximal and distal nerve ends using 10-0 vicrylsutures (Ethicon, Somerville, N.J.). The muscle was drawn back togetherwith 5-0 chromic gut sutures (Ethicon), and the skin was closed withwound clips (Becton Dickinson, Sparks, Md.). Surgical methods were inaccordance with regulations established by the National Research Councilin the Guide for the Care and Use of Laboratory Animals.

Immunogenicity of Grafts Evaluated with Histology.

The grafts were harvested 28 days after implantation. Each animal wasre-anesthetized, and the nerve graft was exposed. Prior to harvesting,the graft was fixed for 1 minute with 3% glutaraldehyde/4%paraformaldehyde in phosphate buffered saline (PBS). Then, the sciaticnerve was transected 5 mm above and below the graft, the distal end wasmarked with a stitch, and the graft was placed in fixative at 4° C.After 30 minutes, the graft was transferred to PBS and stored at 4° C.until it was embedded in paraffin.

Histology was used to inspect the allografts for signs of immunologicalrejection. The tissue was dehydrated with graded alcohol solutions andxylene, and then embedded in paraffin. Longitudinal sections of tissue 7μm thick were cut with a microtome and captured on glass slides.Immunostaining was performed with anti-CD8a+ (Pharmingen, San Diego,Calif.) and anti-macrophage (Chemicon, Temecula, Calif.) primaryantibodies. Horseradish peroxidase (HRP) tagged secondary antibodies,3,3′ diaminobenzidine (DAB) substrate (Vector Labs, Burlingame, Calif.),and an eosin counterstain were used to visualize the invading cells. Thestained sections were visualized on an Olympus IX70 (Melville, N.Y.)inverted microscope, and the images were captured with an OptronicsMagnaFire (Goleta, Calif.) digital color camera. After capturing imagesof the stained tissue sections, the images were combined in AdobePhotoshop to create a composite of the entire graft. Using Scion Imagesoftware (Scion Corporation, Frederick, Mass.), the percentage of areaof the graft covered with positively stained CD8+ and macrophage cellswas determined.

Acellular Graft Models Compared In Vivo.

To study the impact of cellular debris and structural preservation onregeneration, three acellular graft models were examined in vivo.Optimized acellular grafts, grafts created with the Sondell protocol,and freeze/thaw grafts were created as described in the Materials andMethods section. Fresh grafts were also included in the experiments as apositive control. Fresh isografts are a mimic of the clinical autograft.The optimized acellular grafts and Sondell grafts were prepared within30 days of implantation. The time between harvest and implantation ofthe freeze/thaw grafts and fresh grafts was never longer than 30minutes. Donor and host animals were HSD rats.

Histological Comparison of Decellularized Tissues.

A comparison of the ECM structure in the acellular grafts prior toimplantation was conducted by visualizing the basal laminae. The graftswere prepared as previously described, but were never surgicallyimplanted or fixed. The grafts were embedded and cross-sectioned. Ananti-laminin primary antibody (Developmental Studies Hybridoma Bank,Iowa City, Iowa) and a TRITC-conjugated goat anti-mouse secondaryantibody (Jackson ImmunoResearch, West Grove, Pa.) were employed in theimmunostaining procedure.

Regenerative Capacity of Grafts Evaluated with Histology.

As summarized in Table 3, animals with each of the four grafts used inthe acellular model comparison were harvested 28 days afterimplantation. The remaining animals were used to evaluate regenerationafter 84 days. Prior to the 84-day time point, however, seven animalswere put down due to automutilation of their toes (n=1 fresh graft, n=2freeze/thaw grafts, n=1 Sondell grafts, n=3 optimized acellular grafts).To evaluate the regenerative potential of the three acellular graftmodels, longitudinal tissue sections were stained for regenerated axonsusing an anti-neurofilament primary antibody called RT97 (DevelopmentalStudies Hybridoma Bank), an HRP-conjugated secondary antibody, and DAB.Subsequently, cross sections were cut from the midpoint of the graftsand stained for neurofilaments. The stained sections were visualizedwith a 20× objective and images were captured with the digital camera. A20 cm×16 cm image was printed for each sample. The number of nervefibers in each image was counted, and the area of nerve cable in theimage was measured. Because a portion of the nerve cable had beenremoved by sectioning the tissue longitudinally prior to takingcross-sections, the total number of axons in each nerve cable could notbe determined. Instead, axon density was calculated by dividing thenumber of nerve fibers by the area of the cable from which the count wastaken. Select specimens were not used in the axon density analysisbecause less than 33% of the nerve cable remained after the sample wassectioned longitudinally. The number of samples analyzed for each graftand time point is reported with the axon density data. Regions ofconnective tissue at the periphery of the graft, based on morphologicalevaluation, were excluded from the analysis.

TABLE 3 Implants to Evaluate the Regenerative Capacity of OptimizedGrafts Usable Usable Unusable Unusable Put Cross- Cross- Cross- Cross-Total Down Harvested Harvested Sections Sections Sections Sections GraftType Implants Early (28 days) (84 days) (28 days) (84 days) (28 days)(84 days) Fresh 15 0 9 6 8 5 1 1 Sondell 11 0 6 5 6 3 0 2 Freeze/Thaw 100 6 4 6 3 0 1 Optimized 18 3 9 6 7 5 2 1 Acellular

Statistical Analysis.

The Student's t-Test was performed to determine the statisticalsignificance of the differences between results. A significance level ofp<0.05 was used as the cutoff (i.e., p values are reported only forcases in which p<0.05).

Using the present invention the native, cell-free tissue replacementgrafts were immunologically tolerated. To evaluate the immunologicalresponse by a host to the optimized acellular grafts, four experimentalconditions were tested with sciatic nerve graft implants (Table 2). Afresh isograft was used to establish the inflammatory and immuneresponse that results from surgery alone. This served to mimic thecurrent clinical approach (i.e., autograft) for nerve repair (positivecontrol). The fresh allograft established the cellular responseassociated with cell-mediated rejection (negative control). Theacellular allografts were used to determine if there was a reduction oravoidance of immunological rejection by decellularizing the grafts.Finally, acellular isografts were used to determine the immune responsethat resulted from the processing technique and any residual chemicalsremaining in the graft after decellularization.

By staining longitudinal sections of the grafts for cytotoxic T-cellsand macrophages, the level of cell-mediated immune response wasdetermined. CD8+ cytotoxic T-cells were visualized by staining for CD8cell surface markers. Elevated levels of cytotoxic T-cells are expectedin tissues undergoing cell-mediated rejection. Increased levels ofmacrophage cells are also expected in rejected allografts. However,macrophages are also recruited during Wallerian degeneration to cleardebris and release neurotrophic factors for regenerating nerves. At 28days, both cell types could be seen throughout the full length of allthe grafts (FIGS. 4, 7).

FIGS. 4A, 4B, 4C and 4D are longitudinal sections of tissue that werecut from (4A) fresh isografts, (4B) fresh allografts, (4C) optimizedacellular isografts, and (4D) optimized acellular allografts that wereharvested 28 days after implantation. The tissue sections were stainedfor CD8, a surface marker on cytotoxic T-cells. The level of staining inthe fresh allografts was visibly higher, but the optimized acellulargrafts appeared indistinguishable from the fresh isografts. Scalebar=200 μm.

FIG. 5 is a graph that summarizes the cell-mediated immune response inthe fresh and acellular nerve grafts was evaluated by determining thepercentage of tissue covered by CD8+ cells. The infiltration of CD8+cells into the fresh allografts was higher than into the fresh isografts(p<0.01) and the acellular grafts (p<0.005). Fresh allograftsdemonstrated a statistically significant elevation in CD8+ cells. Theoptimized acellular isografts and allografts were statisticallyindistinguishable from the fresh isografts, indicating that after 28days, cell-mediated immune rejection was only occurring in the freshallografts.

FIGS. 6A, 6B, 6C and 6D are longitudinal sections of tissue were cutfrom (6A) fresh isografts, (6B) fresh allografts, (6C) optimizedacellular isografts and (6D) optimized acellular allografts that wereharvested 28 days after implantation. The tissue sections were stainedfor macrophages, immune cells involved in Wallerian degeneration, nerveregeneration, and tissue inflammation. Scale bar=200 μm.

Meanwhile, the levels of CD8+ cells in the acellular isografts andallografts were lower than those observed in the fresh isografts(p<0.05). Macrophage invasion into the fresh isografts was lower thaninto the fresh allografts (p<0.05), but the differences between othergrafts were not statistically significant.

FIG. 7 is a graph that summarizes the level of macrophages present infresh and acellular nerve grafts after 28 days was evaluated bydetermining the percentage of area stained in longitudinal tissuesections. Fresh allografts demonstrated a statistically significantelevation in macrophages compared to fresh isografts. The optimizedacellular isografts and allografts were statistically indistinguishablefrom the fresh isografts, the fresh allografts, and each other,suggesting that any residual chemicals in the graft did not cause asignificant inflammatory response. Thus, histological examination of thelevels of CD8+ cells and macrophages that infiltrated the acellulargrafts suggested that the decellularization process avertedcell-mediated rejection of the grafts.

Optimized Grafts Support Regenerating Axons.

The capacity of the optimized acellular graft to support nerveregeneration was tested by examining the growth of axons through thevarious nerve isografts after 28 and 84 days. All the grafts wereisografts, harvested from and implanted into HSD rats. The grafts thatwere tested included: (1) fresh isografts, (2) optimized acellulargrafts, (3) grafts created with the Sondell protocol, and (4)freeze/thaw grafts. This was accomplished by staining longitudinalsections and cross-sections of the grafts for neurofilaments (i.e.,cytoskeletal proteins found in axons). At 28 days, new axons had growncompletely across the grafts.

FIGS. 8A, 8B and 8C show the Axonal regeneration through acellular nervegrafts was demonstrated by staining longitudinal tissue segments forneurofilaments. The random patterns in the axons at the junctions of theproximal nerve and graft (8A) and the graft and distal nerve (8C)suggest a lack of guidance as the axons crossed into and out of thegraft. However, at the midpoint of the graft (8B) the axons were highlyaligned, suggesting that they were guided by the extracellular structureof the graft. Suture marks (S) at the nerve/graft junction can be seenin images A and C. Scale bar=100 μm.

The axons appeared to meet some resistance in crossing from the proximalnerve end into the graft and from the graft into the distal nerve end,as demonstrated by the non-linearity in neurofilaments around the suturepoints on both ends of the graft (FIGS. 8A, 8C). However, once the axonsextended into graft, they appeared to grow linearly, as demonstrated bythe parallel neurofilaments at the midpoint of the graft (FIG. 8B).Similarly, once the axons found their way into the distal end of thegraft, they appeared to grow linearly in the distal direction (data notshown). The same basic pattern was observed in the 84-day acellularnerve grafts. Thus, the optimized acellular nerve grafts supportedaxonal regeneration and guided the axons toward the distal nerve end.

Optimized Decellularization Process Preserves the ECM.

Images of tissue sections stained for laminin allow for the comparisonof basal laminae preservation by the decellularization protocols. FIGS.9A, 9B, 9C and 9D are cross-sections of basal laminae were visualized bythe staining of laminin protein. The ring-like appearance of the opentubes in (9A) fresh nerve tissue, (9B) tissue decellularized with theoptimized protocol, and (9C) tissue decellularized with the freeze/thawprotocol suggest the preservation of the basal laminae. Rings aredifficult to distinguish in (9D) the Sondell treated tissue, suggestingthat the basal laminae were damaged during the decellularizationtreatment. Scale bar=10 μm.

The ring-like structures in native nerve tissue are open columns ofbasal laminae (FIG. 9A), and similar structures are apparent in thetissue treated with the optimized decellularization protocol (FIG. 9B)and the freeze/thaw protocol (FIG. 9C). In the tissue created with theSondell treatment (FIG. 9D), the basal laminae appear highly fragmented.

Regenerative Capacity of Optimized Graft Surpasses other AcellularModels.

In addition to visually examining the growth of axons through thegrafts, axon density at the midpoint of the grafts was determined. Thesame acellular and fresh isografts that were harvested 28 and 84 daysafter implantation and sectioned longitudinally were subsequentlycross-sectioned at the midpoint of the grafts. The sections were thenstained for neurofilaments and the axon density in each section wasdetermined. In the 28 day grafts, the fresh grafts (n=9) and optimizedacellular grafts (n=7) were nearly identical with axon densities of 0.9and 0.98 axons/100 μm2, respectively.

FIGS. 10A and 10B are graphs that show the regenerative capacity of fournerve graft models was evaluated by measuring axon density incross-sections of the grafts (10A) 28 days after implantation and (10B)84 days after implantation. Fresh isografts served as a model for theautograft (positive control). Axon density in the fresh grafts and theoptimized acellular grafts was statistically indistinguishable.Freeze/thaw grafts had the lowest axon density, implying that thepresence of cellular debris may reduce the regenerative capacity of anacellular graft. The Sondell grafts also demonstrated a statisticallylower axon density than the optimal acellular grafts, suggesting thatpreservation of the ECM increased the regenerative capacity of acellulargrafts.

The freeze/thaw grafts (n=5) had 0.50 axons/100 μm2, and the Sondellgrafts (n=6) had 0.69 axons/100 μm2. Axon density in the freeze/thawgrafts was statistically lower than in the fresh grafts and theoptimized acellular grafts (p<<0.01). The axon density in the Sondellgrafts was also statistically lower than in the fresh grafts (p<0.01)and the optimized acellular grafts (p<0.05).

For the grafts harvested after 84 days, the fresh grafts (n=5) andoptimized acellular grafts (n=5) were again not statistically different,with axon densities of 0.73 and 0.92 axons/100 μm2, respectively (FIG.10B). The freeze/thaw grafts (n=3) had 0.10 axons/100 μm2, and theSondell grafts (n=3) had 0.23 axons/100 μm2. Axon density in thefreeze/thaw grafts was statistically lower than in the fresh grafts(p<0.05) and the optimized acellular grafts (p<0.05). The axon densityin the Sondell grafts was not statistically lower than in the freshgrafts, but was statistically lower than in the optimized acellulargrafts (p<0.05).

Since cellular debris is not removed by the freeze/thawdecellularization process and the ECM is damaged by the Sondelldecellularization process, the higher axon densities at 24 and 84 daysin the optimized acellular grafts suggest that removing cellular debrisand preserving the ECM in acellular nerve grafts improve theregenerative capacity of the grafts.

An alternative method for treating severed peripheral nerves is neededto avoid the requirement of multiple surgeries, donor site morbidity,and other drawbacks associated with the autograft. Acellular nervegrafts are derived from donor nerve tissue, so they are composed ofproteins endogenous to nerve tissue. Because of their naturalcomposition and the fact that axons preferentially grow through thebasal lamina tubes found in nerve tissue, acellular nerve grafts exhibitpotential for use as a next generation nerve graft. The presentinventors found that improving the decellularization process to yield abetter-preserved ECM leads to an improvement in regeneration. However,in order for the optimized acellular grafts to be used clinically, theymust also be immunologically tolerated. As discussed hereinabove, amethod of removing the cellular material responsible for immunologicalrejection was developed that also preserved the ECM of nerve tissue. Thegrafts created with the optimized decellularization treatment werestudies in vivo to determine if they are in fact immunologicallytolerated and how well they support nerve regeneration.

Cellular antigens are predominantly responsible for the immunologicalrejection associated with nerve allografts, particularly the antigensassociated with Schwann cells, endothelial cells and macrophages. Theremoval of cellular components from the graft by the optimized protocolwas correlated with the immunological response of a host animal of adifferent strain than the donor animal. The major histocompatibilitycomplex (MHC) of the rat is called RT1 and is highly polymorphic. Ratstrains can be characterized by their RT1 haplotype (e.g., RT1b, RTd,RT1l). Matching of haplotypes plays a predominant role in allograftsurvival. Gulati, et al., demonstrated that in allografts involvingstains of different RT1 haplotypes, the increased presence of immunecells associated with rejection was readily detectable at 28 days. Thus,fresh nerve tissue from an HSD rat (RT1 b) implanted into a Lewis rat(RT1 l) (i.e., a fresh allograft) should display signs of immunologicalrejection after 28 days. Similarly, an acellular allograft should berejected if the graft contains membrane-bound antigens associated withthe RT1 haplotype.

Rat cytotoxic T-cells carry a CD8 cell surface marker (i.e., they areCD8+ cells), and the presence of cytotoxic T-cells is an importantindicator of cell-mediated graft rejection. However, a moderate numberof CD8+ cells that are not cytotoxic should be present in any nervegraft after 28 days, whether or not it is being rejected. Thenon-cytotoxic CD8+ cells are a subset of macrophages that are known toinvade after sciatic nerve injuries, even in the absence ofimmunological rejection. Macrophages are immune cells that respond tonerve injury, clear cellular debris during nerve degeneration, andsupport regeneration by inducing and producing growth factors. In thecase of a rejected allograft, higher numbers of macrophages should bepresent. However, macrophages also respond to other cues in theregenerating nerve, so an increase in macrophages without a concomitantincrease in CD8+ cells does not indicate rejection. Thus, the presenceof CD8+ and macrophage cells was anticipated in all four grafts beingtested. A statistically significant increase in both CD8+ and macrophagecells in a graft, when compared to a fresh isograft, however, wouldindicate that the graft was undergoing cell-mediated rejection.

It was found that fresh allografts did exhibit a statistical increase inboth CD8+ cells and macrophages, compared to fresh isografts (FIGS. 5and 7). The acellular grafts did not show any increase in CD8+ cells,compared to fresh isografts, indicating that they did not elicitcell-mediated immune rejection. If residual cellular antigens in theacellular grafts were eliciting cell-mediated rejection, the acellularallografts should have demonstrated a higher level of CD8+ cells thanthe acellular isografts. Similarly, the level of macrophages in theacellular allografts should have been higher than in the acellularisografts if the allografts were being rejected. Because the levels ofimmune cells (i.e., both CD8+ cells and macrophages) in the acellularisografts and allografts were similar to one another, the acellulargrafts were not undergoing immunological rejection.

Macrophage invasion into the acellular grafts did appear to be slightlyhigher than that in the fresh isografts, though not significantly. Onepossible cause for the elevation in the level of macrophages in theacellular grafts compared to the fresh isografts is that the open basallamina tubes and the absence of myelin allowed a higher number ofmacrophages to invade and remain inside the acellular grafts. This maybe beneficial since macrophages produce growth factors. In summary, byremoving cellular components from nerve tissue, the antigens that wouldnormally initiate cell-mediated immunological rejection of an allograftwere removed, and the acellular grafts were immunologically toleratedafter 28 days in vivo.

Regenerative Capacity Correlates to Graft Structure and Content.

When engineering the optimized acellular grafts, the two major goalswere to remove cellular material and to provide structural support forregenerating nerves. Accomplishing those two goals led to improvedlevels of regeneration in comparison to the levels seen with otheracellular grafts. The importance of structural support was evident withhistological examination of longitudinal tissue sections. Axons grewlinearly in regions of defined structure (e.g., in the nerve graft anddistal nerve cable), but their path was irregular in the regions wherethe graft was attached to the nerve ends (FIGS. 8A-8C). The irregularpatterns were potentially caused by the misalignment of basal laminae atthe junctions between the nerve ends and the graft. As the axons crossedinto and out of the graft, they had to find new basal laminae to providethem with guidance.

In addition to providing guidance, the optimized acellular grafts alsosupported higher axon densities after 24 and 84 days than the otherpublished acellular graft models (FIGS. 10A and 10B). The lowest axondensities were found in the freeze/thaw grafts. While the structuralpreservation in the freeze/thaw grafts was similar to that in theoptimized grafts (FIGS. 9A-9D), the freeze/thaw procedure was the onlydecellularization procedure that did not wash dead cells out of thegrafts. Thus, a correlation was suggested between the presence of celldebris and a reduction in the level of nerve regeneration. The primarydifference between the Sondell protocol and the optimizeddecellularization protocol was preservation of the ECM (FIGS. 9A-9D).Consequently, the higher axon density in the optimized acellular graftssuggests that providing regenerating axons with an ECM structure thatmimics that of native nerve is important for maximizing regeneration inan acellular graft. The importance of these factors appears to becomemore evident over longer time periods, with the optimized graftdemonstrating axon densities 910% higher than the freeze/thaw graft and401% higher than the Sondell graft after 84 days.

Since fresh isografts were the only grafts that contained living cells(e.g., Schwann cells and macrophages) that aid regeneration, they wereexpected to support higher axon densities than any of the acellulargrafts. However, the data suggest that in 10 mm nerve grafts, thecombination of desirable structure and the removal of cellular debriswere sufficient to attain axon densities statistically indistinguishablefrom those in fresh isografts (FIGS. 10A and 10B). In the case of longergrafts, however, the need for support cells will probably become morecrucial. To address injuries with longer gaps, cells (e.g., Schwanncells) may be incorporated into the optimized acellular grafts prior toimplantation.

Because the optimized acellular graft disclosed herein is a simplersystem than the autograft, it serves as a tool to study individual cellsor growth factors by selectively incorporating them into the graft. Thenatural structural environment of this acellular graft makes it an idealmodel for conducting such experiments. As more information is gainedabout the role of the ECM, support cells, and growth factors, bettertherapeutic systems may be engineered for stimulating nerve regenerationand an off-the-shelf replacement for the autograft can be developed,possibly using this graft as the structural foundation.

While specific alternatives to steps of the invention have beendescribed herein, additional alternatives not specifically disclosed,but known within the art, are intended to fall within the scope of thepresent invention. Thus it is understood that other applications of thepresent invention will be apparent to those skilled in the art upon thereading of the described embodiments and a consideration of the claimsand drawings.

REFERENCES

-   1. Lundborg, G. (2000) A 25-year perspective of peripheral nerve    surgery: evolving neuroscientific concepts and clinical    significance. J. Hand Surg [Am] 25, 391.-   2. Hudson, T. W., Evans, G. R., and Schmidt, C. E. (2000)    Engineering strategies for peripheral nerve repair. Orthop Clin    North Am 31, 485.-   3. Strauch, B. (2000) Use of nerve conduits in peripheral nerve    repair. Hand Clin 16, 123.-   4. Gulati, A. K., Rai, D. R., and Ali, A. M. (1995) The influence of    cultured Schwann cells on regeneration through acellular basal    lamina grafts. Brain Res 705, 118.-   5. Sorensen, J., Fugleholm, K., Moldovan, M., Schmalbruch, H., and    Krarup, C. (2001) Axonal elongation through long acellular nerve    segments depends on recruitment of phagocytic cells from the    near-nerve environment. Electrophysiological and morphological    studies in the cat. Brain Res 903, 185.-   6. Frostick, S. P., Yin, Q., and Kemp, G. J. (1998) Schwann cells,    neurotrophic factors, and peripheral nerve regeneration.    Microsurgery 18, 397.-   7. Ide, C., Osawa, T., and Tohyama, K. (1990) Nerve regeneration    through allogeneic nerve grafts, with special reference to the role    of the Schwann cell basal lamina. Prog Neurobiol 34, 1.-   8. Martini, R. (1994) Expression and functional roles of neural cell    surface molecules and extracellular matrix components during    development and regeneration of peripheral nerves. J Neurocytol 23,    1.-   9. Fawcett, J. W., and Keynes, R. J. (1990) Peripheral nerve    regeneration. Annu Rev Neurosci 13, 43.-   10. Ide, C., et al. (1998) Long acellular nerve transplants for    allogeneic grafting and the effects of basic fibroblast growth    factor on the growth of regenerating axons in dogs: a preliminary    report. Exp Neurol 154, 99.-   11. Sondell, M., Lundborg, G., and Kanje, M. (1998) Regeneration of    the rat sciatic nerve into allografts made acellular through    chemical extraction. Brain Res 795, 44.-   12. Gulati, A. K., and Cole, G. P. (1994) Immunogenicity and    regenerative potential of acellular nerve allografts to repair    peripheral nerve in rats and rabbits. Acta Neurochir (Wien) 126,    158.-   13. Zalewski, A. A., and Gulati, A. K. (1982) Evaluation of    histocompatibility as a factor in the repair of nerve with a frozen    nerve allograft. J Neurosurg 56, 550.-   14. Danielsen, N., Kerns, J. M., Holmquist, B., Zhao, Q., Lundborg,    G., and Kanje, M. (1995) Predegeneration enhances regeneration into    acellular nerve grafts. Brain Res 681, 105.-   15. Osawa, T., Tohyama, K., and Ide, C. (1990) Allogeneic nerve    grafts in the rat, with special reference to the role of Schwann    cell basal laminae in nerve regeneration. J Neurocytol 19, 833.-   16. Pollard, J. D., and Fitzpatrick, L. (1973) An ultrastructural    comparison of peripheral nerve allografts and autografts. Acta    Neuropathol (Berl) 23, 152.-   17. Hall, S. M. (1986) Regeneration in cellular and acellular    autografts in the peripheral nervous system. Neuropathol Appl    Neurobiol 12, 27.-   18. Krekoski, C. A., Neubauer, D., Zuo, J., and Muir, D. (2001)    Axonal regeneration into acellular nerve grafts is enhanced by    degradation of chondroitin sulfate proteoglycan. J Neurosci 21,    6206.-   19. Johnson, P. C., Duhamel, R. C., Meezan, E., and    Brendel, K. (1982) Preparation of cell-free extracellular matrix    from human peripheral nerve. Muscle Nerve 5, 335.-   20. Gulati, A. K., and Cole, G. P. (1990) Nerve graft immunogenicity    as a factor determining axonal regeneration in the rat. J Neurosurg    72, 114.-   21. Gulati, A. K. (1988) Evaluation of acellular and cellular nerve    grafts in repair of rat peripheral nerve. J Neurosurg 68, 117.-   22. National Center for Health Statistics Based on Classification of    Diseases, 9th Revision, Clinical Modifications for the Following    Categories: ICD-9 CM Codes 04.3, 04.5, 04.6, 04.7.-   23. Pollard, J. D., and McLeod, J. G. (1981) Fresh and predegenerate    nerve allografts and isografts in trembler mice. Muscle Nerve 4,    274.-   24. Gulati, A. K. (1998) Immune response and neurotrophic factor    interactions in peripheral nerve transplants. Acta Haematol 99, 171.-   25. Guenther, E., Stark, O. (1977) The major histocompatibility    system of the rat (Ag—B or H-1 system), in: D. Goetze, ed. The major    histocompatibility system in man and animals. Springer-Verlag, New    Yok, pp. 207-253.-   26. Jander, S., Lausberg, F., and Stoll, G. (2001) Differential    recruitment of CD8+ macrophages during Wallerian degeneration in the    peripheral and central nervous system. Brain Pathol 11, 27.-   27. Hirata, K., Mitoma, H., Ueno, N., He, J. W., and    Kawabuchi, M. (1999) Differential response of macrophage    subpopulations to myelin degradation in the injured rat sciatic    nerve. J Neurocytol 28, 685.-   28. Bruck, W. (1997) The role of macrophages in Wallerian    degeneration. Brain Pathol 7, 741.-   29. Perry, V. H., and Brown, M. C. (1992) Macrophages and nerve    regeneration. Curr Opin Neurobiol 2, 679.-   30. Pollard, J. D., and Fitzpatrick, L. (1973) A comparison of the    effects of irradiation and immunosuppressive agents on regeneration    through peripheral nerve allografts: an ultrastructural study. Acta    Neuropathol (Berl) 23, 166.

1-40. (canceled)
 41. A method for preparing a native, acellular tissuereplacement comprising the steps of: obtaining a tissue; soaking thetissue for at least six hours in a solution comprising one or moresulfobetaines; treating the tissue in a mixture of one or moresulfobetaines and an anionic surface-active detergent; and washing thetissue in one or more solutions of a buffered salt to remove the excessanionic surface-active detergent to form the native, acellular tissuereplacement that is immunologically tolerated when implanted.
 42. Themethod of claim 41, further comprising the step of storing the native,acellular tissue replacement in a buffered salt solution until needed.43. The method of claim 41, wherein the sulfobetaines have hydrophilictails of 10 to 16 carbons.
 44. The method of claim 41, furthercomprising the step of adhering one or more bioactive agents to thetissue replacement.
 45. The method of claim 41, wherein the native,acellular tissue comprise mammalian tissue except collagen and muscletissue.
 46. The method of claim 44, wherein the one or more bioactivecompounds comprises a drug.
 47. The method of claim 41, wherein thenative, acellular tissue comprises nerve tissue.
 48. The method of claim41, wherein the native, acellular tissue replacement further comprises astructure selected from the group consisting of a tube, a sheet, a film,a scaffold, and a tissue transplant for delivery into the body.
 49. Themethod of claim 41, wherein the sulfobetaine comprises SB-16.
 50. Themethod of claim 41, wherein the anionic surface-active detergent isselected from the group consisting of sodium deoxycholate, Triton X-200and dodecylbenzene sulfonate.
 51. The method of claim 41, wherein thestep of washing the tissue comprises one or more washes in a bufferedsalt solution comprising 100 mM sodium and 50 mM phosphate for at least15 minutes each.
 52. The method of claim 45, wherein the tissue isharvested from a mammalian cadaver.
 53. The method of claim 52, whereinthe tissue is cleaned of fat and blood after harvesting and rinsed twoor more times in deionized distilled water.
 54. A native, acellulartissue replacement made by the method of claim
 41. 55. A kit for tissuereplacement comprising the native, acellular tissue replacement of claim54.
 56. The kit of claim 55, wherein the native, acellular tissuereplacement further comprises a tube, a sheet, a film, a scaffold, or atissue transplant.
 57. The kit of claim 56, wherein the native,acellular tissue replacement further comprises a polymer, a bioactivecompound or combinations thereof.
 58. The kit of claim 56, furthercomprising a sheet of instructions for use of the native, acellulartissue replacement.
 59. A method for preparing a native, acellular nervetissue replacement comprising the steps of: obtaining a nerve tissue;soaking the tissue for at least six hours in a solution comprising oneor more sulfobetaines; treating the nerve tissue in a mixture of one ormore sulfobetaines and an anionic surface-active detergent; and washingthe nerve tissue in one or more solutions of a buffered salt to removeexcess detergent to form the native, acellular tissue replacement,wherein the basal laminae and endoneurium layer substantially retain thenative extracellular matrix structure.
 60. The method of claim 59,wherein the native, acellular nerve tissue replacement, when implanted,elicits a T-cell mediated immune response that is less than an immuneresponse triggered by an allogeneic implant.
 61. The method of claim 59,wherein the native, acellular nerve tissue replacement allows for higheraxon density when implanted relative to an untreated nerve tissue graft.62. A method of making a native, acellular tissue replacementcomprising: obtaining a tissue; soaking the tissue for at least sixhours in a solution comprising one or more sulfobetaines; treating thetissue in a mixture of one or more sulfobetaines and an anionicsurface-active detergent; and washing the tissue in one or moresolutions of a buffered salt to remove the excess anionic surface-activedetergent to form the native, acellular tissue replacement, wherein thenative, acellular tissue replacement is immunologically tolerated whenimplanted into a mammal.